The spread of tumor cells and the formation of distant metastasis remain the main causes of mortality in cancer patients. However, the mechanisms governing the release of cells from micro-environmental constraints remain unclear. E-cadherin negatively controls the invasion of epithelial cells by maintaining cell–cell contacts. Furthermore, the inactivation of E-cadherin triggers invasion in vitro. However, the role of E-cadherin is complex, as metastasizing cells maintain E-cadherin expression, which appears to have a positive role in the survival of tumor cells. In this report, we present a novel mechanism delineating how E-cadherin function is modulated to promote invasion. We have previously shown that E-cadherin is associated with p100AmotL2, which is required for radial actin formation and the transmission of mechanical force. Here, we present evidence that p60AmotL2, which is expressed in invading tumor cells, binds to the p100AmotL2 isoform and uncouples the mechanical constraint of radial actin filaments. We show for the first time that the coupling of E-cadherin to the actin cytoskeleton via p100AmotL2 is directly connected to the nuclear membrane. The expression of p60AmotL2 inactivates this connection and alters the properties of the nuclear lamina, potentiating the invasion of cells into micropores of the extracellular matrix. In summary, we propose that the balance of the two AmotL2 isoforms is important in the modulation of E-cadherin function and that an imbalance of this axis promotes ameboid cell invasion.

Stiffness and actomyosin contractility are intrinsic mechanical properties of animal cells required for the shaping of tissues. However, whether tissue stem cells (SCs) and progenitors located within SC niche have different mechanical properties that modulate their size and function remains unclear. Here, we show that hair follicle SCs in the bulge are stiff with high actomyosin contractility and resistant to size change, whereas hair germ (HG) progenitors are soft and periodically enlarge and contract during quiescence. During activation of hair follicle growth, HGs reduce contraction and more frequently enlarge, a process that is associated with weakening of the actomyosin network, nuclear YAP accumulation, and cell cycle reentry. Induction of miR-205 , a novel regulator of the actomyosin cytoskeleton, reduces actomyosin contractility and activates hair regeneration in young and old mice. This study reveals the control of tissue SC size and activities by spatiotemporally compartmentalized mechanical properties and demonstrates the possibility to stimulate tissue regeneration by fine-tuning cell mechanics.

Vinculin, a mechanotransducer associated with both adherens junctions (AJ) and focal adhesions (FA) plays a central role in force transmission through these cell-cell and cell-substratum contacts. Here we describe the conditional knock out (KO) of vinculin in murine skin. Remarkably, we find that the loss of vinculin function results in the loss of bulge stem cell (BuSC) quiescence. We demonstrate that vinculin KO cells are impaired in force generation resulting in mechanically weak AJs. Mechanistically, vinculin functions by keeping α-catenin in a stretched conformation, which in turn regulates the retention of YAP1, another potent mechanotransducer and regulator of cell proliferation, to the junctions. Conditional KO of α-catenin specifically in the BuSCs further corroborates the importance of stable AJs in the maintenance of quiescence and stemness. Altogether, our data provides definitive mechanistic insights into the hitherto unexplored regulatory link between the mechanical stability of cell-junctions and the maintenance of BuSC quiescence.

Mechanically stable specific heterodimerization between small protein domains have a wide scope of applications, from using as a molecular anchorage in single-molecule force spectroscopy studies of protein mechanics, to serving as force-bearing protein linker for modulation of mechanotransduction of cells, and potentially acting as a molecular crosslinker for functional materials. Here, we explore the possibility to develop heterodimerization system with a range of mechanical stability from a set of recently engineered helix-heterotetramers whose mechanical properties have yet to be characterized. We demonstrate this possibility using two randomly chosen helix-heterotetramers, showing that their mechanical properties can be modulated by changing the stretching geometry and the number of interacting helices. These helix-heterotetramers and their derivatives are sufficiently stable over physiological temperature range. Using it as mechanically stable anchorage, we demonstrate the applications in single-molecule manipulation studies of the temperature dependent unfolding and refolding of a titin immunoglobulin domain and α-actinin spectrin repeats.

Metastasis is widely accepted to be responsible for approximately 90% of all cancer deaths. Current research on metastasis prediction often centers on gene sequencing; however, these analyses must account for the complexity of gene regulation and rely on comprehensive datasets. To investigate the process from a simpler, non-genomic angle, some studies indicate differences in cell adhesion force, an important physical process in metastasizing cells. However, cell adhesion force methods tend to focus on cell population approaches and therefore have their drawbacks in cost or efficiency, rendering them impractical outside a research setting. In this work, we test a novel and inexpensive bead-pipette assay to investigate the adhesion forces of non-metastatic NIH3T3 cells and mutated RasV12 cells, a metastatic model cell line. Control cells and RasV12 cells were evaluated with wound healing, spreading area, and focal adhesion (FA) analysis assays. Then cells were tested by the novel bead-pipette assay, which uses a fibronectin-coated bead and a glass micropipette to measure cell adhesion force using Hooke’s law. The RasV12 cells had faster migration, polarized cell shape, and smaller FA area than control cells. The RasV12 cells also exerted higher adhesion forces than control cells and a potential force threshold was determined for distinguishing metastatic cells through a Receiver Operating Characteristic (ROC) curve. An ROC curve was computed for all other assays and the bead-pipette assay was shown to perform higher as a classifier than other assays. The RasV12 cells had increased metastatic potential compared to control. The novel bead-pipette assay showed potential as a classifier for determining metastasizing cells from non-metastatic cells. With further work, it may serve as a clinical diagnostic tool for cancer patients or as a testbed to be used in the development of anti-metastatic drugs.

Mechanical loading plays an important role in bone homeostasis. However, molecular mechanisms behind the mechanical regulation of bone homeostasis are poorly understood. We previously reported p130Cas (Cas) as a key molecule in cellular mechanosensing at focal adhesions. Here, we demonstrate that Cas is distributed in the nucleus and supports mechanical loading-mediated bone homeostasis by alleviating NF-κB activity, which would otherwise prompt inflammatory processes. Mechanical unloading modulates Cas distribution and NF-κB activity in osteocytes, the mechanosensory cells in bones. Cas deficiency in osteocytes increases osteoclastic bone resorption associated with NF-κB-mediated RANKL expression, leading to osteopenia. Upon shear stress application on cultured osteocytes, Cas translocates into the nucleus and down-regulates NF-κB activity. Collectively, fluid shear stress-dependent Cas-mediated alleviation of NF-κB activity supports bone homeostasis. Given the ubiquitous expression of Cas and NF-κB together with systemic distribution of interstitial fluid, the Cas-NF-κB interplay may also underpin regulatory mechanisms in other tissues and organs.

Dystrophin is the largest protein isoform (427 kDa) expressed from the gene defective in Duchenne muscular dystrophy, a lethal muscle-wasting and genetically inherited disease. Dystrophin, localized within a cytoplasmic lattice termed costameres, connects the intracellular cytoskeleton of a myofiber through the cell membrane (sarcolemma) to the surrounding extracellular matrix. In spite of its mechanical regulation roles in stabilizing the sarcolemma during muscle contraction, the underlying molecular mechanism is still elusive. Here, we systematically investigated the mechanical stability and kinetics of the force-bearing central domain of human dystrophin that contains 24 spectrin repeats using magnetic tweezers. We show that the stochastic unfolding and refolding of central domain of dystrophin is able to keep the forces below 25 pN over a significant length change up to ∼800 nm in physiological level of pulling speeds. These results suggest that dystrophin may serve as a molecular shock absorber that defines the physiological level of force in the dystrophin-mediated force-transmission pathway during muscle contraction/stretch, thereby stabilizing the sarcolemma.

Extracellular vesicles (EVs) play an important role as active messengers in intercellular communication and distant microenvironment modeling. Increasingly, these EVs are recognized as important biomarkers for clinical diagnostics. However, current isolation methods of EVs are time-consuming and ineffective due to the high diffusive characteristics of nanoparticles coupled with fluid flow instability. Here, we develop a microfluidic CEntrifugal Nanoparticles Separation and Extraction (µCENSE) platform for the rapid and label-free isolation of microvesicles. By utilizing centrifugal microhydrodynamics, we subject the nanosuspensions between 100 nm and 1000 nm to a unique fluid flow resulting in a zonal separation into different outlets for easy post-processing. Our centrifugal platform utilizes a gentle and efficient size-based separation without the requirements of syringe pump and other accessories. Based on our results, we report a high separation efficiency of 90% and an extraction purity of 85% within a single platform. Importantly, we demonstrate high EV extraction using a table top centrifuge within a short duration of eight minutes. The simple processes and the small volume requirement further enhance the utility of the platform. With this platform, it serves as a potential for liquid biopsy extraction and point-of-care diagnostics.

Formins, an important family of force-bearing actin-polymerizing factors, function as homodimers that bind with the barbed end of actin filaments through a ring-like structure assembled from dimerized FH2 domains. It has been hypothesized that force applied to formin may facilitate transition of the FH2 ring from an inhibitory closed conformation to a permissive open conformation, speeding up actin polymerization. We confirm this hypothesis for mDia1 dependent actin polymerization by stretching a single-actin filament in the absence of profilin using magnetic tweezers, and observe that increasing force from 0.5 to 10 pN can drastically speed up the actin polymerization rate. Further, we find that this force-promoted actin polymerization requires torsionally unconstrained actin filament, suggesting that mDia1 also senses torque. As actin filaments are subject to complex mechanical constraints in living cells, these results provide important insights into how formin senses these mechanical constraints and regulates actin organization accordingly.

Cell migration is a highly dynamic process that plays pivotal roles in both physiological and pathological processes. We have previously reported that p130Cas supports cell migration through the binding to Src as well as phosphorylation-dependent association with actin retrograde flow at focal adhesions. However, it remains elusive how phosphorylated Cas interacts with actin cytoskeletons. We observe that the actin-binding protein, tensin 1, co-localizes with Cas, but not with its phosphorylation-defective mutant, at focal adhesions in leading regions of migrating cells. While a truncation mutant of tensin 1 that lacks the phosphotyrosine-binding PTB and SH2 domains (tensin 1-SH2PTB) poorly co-localizes or co-immunoprecitates with Cas, bacterially expressed recombinant tensin 1-SH2PTB protein binds to Casin vitroin a Cas phosphorylation-dependent manner. Furthermore, exogenous expression of tensin 1-SH2PTB, which is devoid of the actin-interacting motifs, interferes with the Cas-driven cell migration, slows down the inward flux of Cas molecules, and impedes the displacement of Cas molecules from focal adhesions. Taken together, our results show that tensin 1 links inwardly moving actin cytoskeletons to phosphorylated Cas at focal adhesions, thereby driving cell migration.